GAD 67KD antisense in colon cancer cells inhibits cell growth and sensitizes to butyrate and pH reduction and H2O2 and gamma-radiation

In eukaryotes, glutamate decarboxylase (GAD) expression was found in brain, kidney, and several kinds of tumor tissues. But its function has been emphasized only as a neurotransmitter-synthesizer, the role in controlling intracellular physiology is poorly understood. According to our studies, when GAD 67KD expression in colon cancer HT-29 cell was repressed by antisense DNA, the cell proliferation was significantly inhibited. GAD 67KD antisensed cells exhibited the low glutathione and high reactive oxygen species level. More importantly, these cells were extremely sensitive to butyrate or pH reduction, both of which naturally cause metabolic stress in the colon lumen, as well as H2O2 and ionizing radiation. These data indicate that GAD 67KD regulates the intracellular redox potential and is important for resistance to acidic or oxidative stress. So, based on these results, we suggest that inhibition of GAD 67KD expression has potentially important implications for overcoming the drug resistance of cancer cells.     

Hetero- and autoprocessing of the extracellular metalloprotease (Mpr) in Bacillus subtilis

Most proteases are synthesized as inactive precursors which are processed by proteolytic cleavage into a mature active form, allowing regulation of their proteolytic activity. The activation of the glutamic-acid-specific extracellular metalloprotease (Mpr) of Bacillus subtilis has been examined. Analysis of Mpr processing in defined protease-deficient mutants by activity assay and Western blotting revealed that the extracellular protease Bpr is required for Mpr processing. pro-Mpr remained a precursor form in bpr-deficient strains, and glutamic-acid-specific proteolytic activity conferred by Mpr was not activated in bpr-deficient strains. Further, purified pro-Mpr was processed to an active form by purified Bpr protease in vitro. We conclude that Mpr is activated by Bpr in vivo, and that heteroprocessing, rather than autoprocessing, is the major mechanism of Mpr processing in vivo. Exchange of glutamic acid for serine in the cleavage site of Mpr (S93E) allowed processing of Mpr into its mature form, regardless of the presence of other extracellular proteases, including Bpr. Thus, a single amino acid change is sufficient to convert the Mpr processing mechanism from heteroprocessing to autoprocessing.     

Differential regulation of IGF-II-induced IL-8 by extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases in human keratinocytes

In order to study the relationship between insulin like growth factor-II (IGF-II) and interleukin-8 (IL-8) that are upregulated in psoriasis, we monitored IL-8 expression in IGF-II-treated human keratinocytes and explored the signaling pathways of IL-8 expression by IGF-II. IGF-II increased the IL-8 mRNA and protein levels in human keratinocytes. The upregulation of IL-8 expression by IGF-II was reduced by pretreatment with inhibitors of tyrosine kinase, Src, PI3-kinase, and ERK, but not by p38. Furthermore, IGF-II remarkably increased the DNA binding activities of NF-kappaB and AP-1, and the IL-8 promoter activity. However, cotransfection with IkappaB mutant blocked the IGF-II-induced IL-8 promoter activity. In addition, cotransfection with dominant negative MEK1 mutant, but not with dominant negative p38 mutant, blocked the IGF-II-induced IL-8 promoter activity. These results suggest that IGF-II is involved in the pathogenesis of psoriasis by inducing IL-8 gene expression through the tyrosine kinase-Src-ERK1/2-AP-1 pathway, and the PI3-kinase and NF-kappaB pathway.     

Molecular dynamics simulations of papilloma virus E2 DNA sequences: dynamical models for oligonucleotide structures in solution

The specificity of papilloma virus E2 protein-DNA binding depends critically upon the sequence of a region of the DNA not in direct contact with the protein, and represents one of the simplest known examples of indirect readout. A detailed characterization of this system in solution is important to the further investigation hypothesis of a structural code for DNA recognition by regulatory proteins. In the crystalline state, the E2 DNA oligonucleotide sequence, d(ACCGAATTCGGT), exhibits three different structural forms. We report herein studies of the structure of E2 DNA in solution based on a series of molecular dynamics (MD) simulations including counterions and water, utilizing both the canonical and various crystallographic structures as initial points of departure. All MDs converged on a single dynamical structure of d(ACCGAATTCGGT) in solution. The predicted structure is in close accord with two of the three crystal structures, and indicates that a significant kink in the double helix at the central ApT step in the other crystal molecule may be a packing effect. The dynamical fine structure was analyzed on the basis of helicoidal parameters. The calculated curvature in the sequence was found to originate primarily from YPR steps in the regions flanking the central AATT tract. In order to study the role of structural adaptation of the DNA in the binding process, a subsequent simulation on the 16-mer cognate sequence d(CAACCGAATTCGGTTG) was initiated from the crystallographic coordinates of the bound DNA in the crystal structure of the protein DNA complex. MD simulations starting with the protein-bound form relaxed rapidly back to the dynamical structure predicted from the previous simulations on the uncomplexed DNA. The MD results show that the bound form E2 DNA is a dynamically unstable structure in the absence of protein, and arises as a consequence of both structural changes intrinsic to the sequence and induced by the interaction with protein.     

The effect of soluble LAG-3 (CD223) treatment in fetal thymic organ culture

Lymphocyte activation gene-3 (LAG-3; CD223) is structurally similar to CD4 and binds to MHC class II with a 100-fold higher affinity than that of CD4. Soluble LAG-3 (sLAG-3Ig) might be useful for immunotherapy by inducing MHC class II-mediated cell activation. A new form of sLAG-3Ig was constructed containing a critical binding site (D1 and D2 region) to MHC class II, combined with a Fc portion of an immunoglobulin gamma1. After treatment of sLAG-3Ig in fetal thymic organ culture from DO11.10 transgenic mouse, CD4(+) T cell precursors were increased in the positive selection but not affected in the negative selection. Further analysis by treating sLAG-3Ig on thymic epithelial cells revealed that CD40 and MHC class II were up-regulated. These results may demonstrate that the treatment of sLAG-3Ig increases the precursor frequency of CD4(+) T cells by activation of thymic epithelial cells.     

Identification and quantification of mutagenic halogenated cytosines by gas chromatography,fast atom bombardment,and electrospray ionization tandem mass spectrometry

Oxidative modification of nucleic acids has been implicated in carcinogenesis. One potential mechanism involves halogenation by the myeloperoxidase and eosinophil peroxidase systems of phagocytes. In the current studies, three mass spectrometric methods for the in vitro and in vivo analysis of halogenated cytosines and deoxycytidines were compared: gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) with a quadrupole instrument, fast atom bombardment or electrospray ionization (ESI) tandem MS with a four-sector magnetic instrument, and liquid chromatography ESI tandem MS (HPLC-ESI-MS/MS) with an ion-trap instrument. GC-EI-MS with selected ion monitoring of dimethyl-tert-butylsilyl derivatives of nucleobases was the most sensitive method. High-energy collisionally induced dissociation MS/MS analysis with a four-sector magnetic instrument yielded detailed structural information about halogenated nucleoside adducts but required relatively large amounts of material. The most sensitive analysis of intact halogenated deoxycytidine was achieved with extracted ion chromatograms using HPLC-ESI-MS/MS with an ion-trap instrument. Our results indicate that GC-EI-MS is the methodology of choice for ultrasensitive analysis of halogenated cytosines. HPLC-ESI-MS/MS provides greater structural detail for these compounds and may rival GC-EI-MS in sensitivity with more advanced liquid chromatography applications. The mass spectrometric methods we have developed should be useful for evaluating the role of phagocyte-derived oxidants in halogenating nucleobases, nucleosides, and DNA at sites of inflammation.     

Isolation and characterization of Neurospora crassa mutants resistant to antifungal plant defensins

Twenty-five Neurospora crassa mutants obtained by chemical mutagenesis were screened for increased resistance to various antifungal plant defensins. Plant defensin-resistant N. crassa mutants were further tested for their cross-resistance towards other families of structurally different antimicrobial peptides. Two N. crassa mutants, termed MUT16 and MUT24, displaying resistance towards all plant defensins tested but not to structurally different antimicrobial peptides were selected for further characterization. MUT16 and MUT24 were more resistant towards plant defensin-induced membrane permeabilization as compared to the N. crassa wild-type. Based on the previously demonstrated key role of fungal sphingolipids in the mechanism of growth inhibition by plant defensins, membrane sphingolipids of MUT16 and MUT24 were analysed. Membranes of these mutants contained structurally different glucosylceramides, novel glycosylinositolphosphorylceramides, and an altered level of steryl glucosides. Evidence is provided to link these clear differences in sphingolipid profiles of N. crassa mutants with their resistance towards different plant defensins.     

Determination of UDP-N-acetylglucosamine:β-D-mannoside-1,4-N-acetylglucosaminyltransferase-III in patients sera with chronic hepatitis and liver cirrhosis using a monoclonal antibody

The glycoprotein UDP-N-acetylglucosamine: beta-D-mannoside-1,4-N-acetylglucosaminyltransferase-III (GnT-III) catalyzes the addition of N-acetylglucosamine via a beta-1, 4-linkage to the beta-linked mannose of the trimannosyl core of N-linked glycans. It has been reported that the expression of GnT-III increases in many oncogenically transformed cells and human hepatocellular carcinoma (HCC) tissues, and GnT-III enzyme activity in serum can be used for the detection and monitoring of primary hepatomas and hepatocellular carcinomas. A solid-phase enzyme-linked immunosorbent sandwich assay in which a polyclonal antibody (PAb) to aglycosylrecombinant GnT-III (AGR-GnT-III) and a monoclonal antibody (mAb) are employed as a capture protein and probe protein, respectively, is described. The sensitivity of the PAb-mAb sandwich assay, as determined by the dose-response effect for AGR-GnT-III, was 10 ng/ml. This assay was specific for GnT-III and did not detect beta-1, 6-N-acetylglucosaminyltrasferase-V (GnT-V). AGR-GnT-III concentrations in 377 serum specimens were determined by the PAb-mAb sandwich assay and the results were analyzed based on the disease category, using 1.99 microg/mL (AGR-GnT-III) as a cut-off value. The AGR-GnT-III level of 61 normal serum samples was 0.57 +/- 0.71 microg/ml (mean +/- SD). The results revealed an elevation in serum AGR-GnT-III levels in 60 of 86 patients (3.03 +/- 2.04 microg/ml) with liver cirrhosis (LC) and 86 of 91 patients (2.73 +/- 0.59 microg/ml) with chronic hepatitis (CH). By contrast, 3 of 61 normal subjects, 9 of 34 patients (1.02 +/- 1.03 microg/ml) with acute hepatitis and 8 of 38 patients (1.79 +/- 0.56 microg/ml) with a variety of non-hepatic diseases exhibited a slight increase above the cut-off value. These results indicate that serum AGR-GnT-III levels are elevated predominantly in LC or CH cases. Serum AGR-GnT-III concentration, as measured by the developed PAb-mAb sandwich assay, may be a useful differential marker as a diagnostic aid for CH and/or LC and warrants further investigations with expanded serum panels.